Diabetic nephropathy therapies

ABSTRACT

The present invention relates to methods and compounds for treating specific early stage aspects and late stage aspects of diabetic nephropathy. Methods and compounds for treating various physiological features associated with early stage and with late stage diabetic nephropathy are also provided.

This application claims the benefit of U.S. Provisional Application Ser.No. 60/544,121, filed on 11 Feb. 2004; U.S. Provisional Application Ser.No. 60/561,018, filed on 8 Apr. 2004; U.S. Provisional Application Ser.No. 60/578,401, filed on 9 Jun. 2004; and U.S. Provisional ApplicationSer. No. 60/620,802, filed on 20 Oct. 2004, each of which isincorporated by reference herein it its entirety.

FIELD OF THE INVENTION

The present invention relates to methods and compounds for treatingspecific early stage aspects and late stage aspects of diabeticnephropathy. Methods and compounds for treating various physiologicalfeatures associated with early stage and with late stage diabeticnephropathy are also provided.

BACKGROUND OF THE INVENTION

A renal disorder is any alteration in normal physiology and function ofthe kidney. Renal disorders can result from a wide range of acute andchronic conditions and events, including physical, chemical, orbiological injury, insult, or trauma, disease, such as, for example,hypertension, diabetes, congestive heart failure, lupus, sickle cellanemia, and various inflammatory and autoimmune diseases, HIV-associatednephropathies, etc. Renal disorders can lead to reduced kidney function,hypertension, and renal failure, seriously compromising quality of life,sometimes requiring dialysis and in certain circumstances, kidneytransplantation.

Diabetic nephropathy is a major long-term complication of diabetesmellitus, and is the leading indication for dialysis and kidneytransplantation in the United States. (Marks and Raskin, 1998, Med ClinNorth Am, 82:877-907.) The development of diabetic nephropathy is seenin 25 to 50% of Type I and Type 2 diabetic individuals. Accordingly,diabetic nephropathy is the most common cause of end-stage renal diseaseand kidney failure in the Western world.

Contributing risk factors associated with the development of diabeticnephropathy (and other renal disorders) in subjects with Type 1 or Type2 diabetes include hyperglycemia, hypertension, altered glomerularhemodynamics, and increased or aberrant expression of various growthfactors, including transforming growth factor-beta (TGFβ), insulin-likegrowth factor (IGF)-I, vascular endothelial growth factor-a (VEGF-A),and connective tissue growth factor (CTGF). (See, e.g., Flyvbjerg (2000)Diabetologia 43:1205-23; Brosius (2003) Exp Diab Res 4:225-233; Gilbertet al. (2003) Diabetes Care 26:2632-2636; and International PublicationNo. WO 00/13706.)

Current treatment strategies directed at slowing the progression ofdiabetic nephropathy using various approaches, including optimizedglycemic control (through modification of diet and/or insulin therapy)and hypertension control, have demonstrated varying degrees of success.For example, both angiotensin-converting enzyme (ACE) inhibitors andangiotensin receptor blockers (ARBs), administered to reducehypertension, have been shown to delay progression or development ofnephropathy and macroalbuminuria. Several clinical trials haveestablished the benefits of ACE inhibitors and ARBs in patients withdiabetes. However, although ACE inhibitors have been shown to delayrenal decline in patients with Type 1 diabetes, the renoprotectiveeffect of these agents in patients with Type 2 diabetes is less clear.(Raij (2003) Am J. Hypertens 16:46S-49S.)

Further, while glycemic and blood pressure control therapiessignificantly decrease the morbidity and mortality associated withdiabetic nephropathy by delaying progression of associated pathologies,such conventional therapies do not adequately halt the progression ofthe disease and thus fail to provide a complete therapeutic effect. Inaddition, administration of ACE inhibitors or ARBs, the current standardof care, are not universally effective and only minimally delay, but donot remove, the need for kidney transplantation.

Other treatment strategies have focused on one or more growth factors astherapeutic targets. Therapies directed at inhibiting VEGF or TGFβ,either alone or in combination with ACE inhibitors or ARBs, have beenexamined. (See, e.g., De Vriese et al. (2001) J. Am Soc Nephrol12:993-1000; Flyvbjerg et al. (2002) Diabetes 51:3090-3094; Ziyadeh etal, (2000) Proc Natl Acad Sci 97:8015-8020; Chen et al. (2003) BiochemBiophys Res Commun 300:16-22; and Benigni et al. (2003) J. Am SocNephrol 14:1816-1824.) Such therapeutic approaches, however, have notprovided amelioration of all aspects of renal pathology (e.g., alteredand impaired renal function and structure) associated with diabeticnephropathy. For example, inhibition of TGFβ as a therapeutic target fordiabetic nephropathy was not effective at attenuating albuminuria indb/db mice, despite the beneficial effects such treatment had onglomerular matrix expansion. (See Ziyadeh et al, supra) In addition,while administration of anti-VEGF antibodies to diabetic db/db miceprovided benefit to diabetes-associated increased permeability in thekidney, only minimal beneficial effects on mesangial expansion wereobserved. (See Flyvbjerg et al (2002), supra.) Therefore, although suchtherapies offer promise, alone or in combination, none has resulted inamelioration of both early (e.g., glomerular hyperfiltration, increasedglomerular filtration rate. Microalbuminuria, etc.) and late (e.g.,decreased glomerular filtration rate, macroalbuminuria, excessivemesangial matrix expansion, etc.) pathological features associated withchronic renal disease, e.g., diabetic nephropathy. Thus, there is a needin the art for a complete therapy for treatment of diabetic nephropathythat ameliorates both early and late stages symptoms and pathologiesassociated with the development and progression of the disease.

In addition to the above deficiencies, current therapies for diabeticnephropathy have limited applicability/efficacy due to lack ofspecificity. In particular, VEGF— or TGFβ-targeted therapies maycompromise the beneficial activities of these growth factors, such asangiogenesis, tumor suppression, and proper immune system development.For example, while TGFβ has been associated with development offibrosis, it is also an important mediator of immune development andtumor suppression, suggesting that inhibition of TGFβ might haveunwanted and potentially adverse secondary effects. Therefore, there isa need in the art for a more selective therapeutic approach for diabeticnephropathy.

In summary, there is an existing need in the art for a therapeuticapproach for treating renal disease, in particular diabetic nephropathy,which is effective at various stages (e.g., early stage and late stagediabetic nephropathy) in the development and progression of the disease.In particular, there is a need for a complete treatment for diabeticnephropathy, one effective in treating both early stage features andlate stage features of diabetic nephropathy such as, for example,hyperfiltration (early stage), increased glomerular permeability (earlystage), increased glomerular filtration rate (early stage),microalbuminuria (early stage), macroalbuminuria (late stage), anddecreased glomerular filtration rate (late stage). There is a need for atherapeutic approach that more completely addresses various and distinctprocesses associated with development and progression of diabeticnephropathy and other renal diseases. In particular, there is a need fortherapies that target both non-fibrotic (e.g., hyperfiltration) andfibrotic (e.g., mesangial matrix expansion) processes associated withdiabetic nephropathy. In addition, there is a need for a therapeuticapproach for treating renal disease in general, and diabetic nephropathyin particular, that provides both structural and functional benefits.

The present invention addresses these needs by identifying the role ofCTGF in various processes associated with the development andprogression of renal disorders such as, e.g., diabetic nephropathy, andby providing methods for inhibiting and preventing these processes. Theinvention further addresses existing needs by providing methods andagents that can be applied to the treatment and prevention of renaldiseases, particularly, renal disease associated with diabetes, and mostparticularly, diabetic nephropathy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows anti-CTGF antibody administration reduced kidney weightincrease in diabetic db/db mice.

FIG. 2 shows anti-CTGF antibody administration reduced creatinineclearance in diabetic db/db mice.

FIG. 3 shows anti-CTGF antibody administration reduced urinary albuminexcretion in diabetic db/db mice.

FIG. 4 shows the correlation between CTGF and VEGF levels in humanvitreous.

FIG. 5 shows anti-CTGF antibody administration reduced urine volume indiabetic db/db mice.

FIG. 6 shows anti-CTGF antibody administration reduced basement membranethickening in kidneys of diabetic db/db mice.

FIG. 7 shows anti-CTGF antibody administration reduced proteinuria in arat model of diabetic nephropathy.

FIG. 8 shows anti-CTGF antibody administration reduced BUN levels in arat model of diabetic nephropathy.

FIG. 9 shows anti-CTGF antibody administration improved glomerularfiltration rate in a rat model of diabetic nephropathy.

SUMMARY OF TH INVENTION

The present invention relates to methods and compounds for treatment orprevention of specific early stage aspects and late stage aspects ofdiabetic nephropathy, and for treatment or prevention of variousphysiological features associated with early stage and with late stagediabetic nephropathy are also provided.

It is specifically contemplated that, in preferred embodiments of eachof the methods described below, the preferred subject is a humansubject.

In one embodiment, the present invention provides a method for reducingcreatinine clearance in a subject having or at risk for having diabetesor early stage diabetic nephropathy, the method comprising administeringto the subject a therapeutically effective amount of an agent thatinhibits CTGF, thereby reducing creatinine clearance in the subject.Normal creatinine clearance levels in humans are typically about 97 to137 ml/min. (adult males) and 88 to 128 ml/min (in adult females).Therefore, methods of reducing creatinine clearance levels to theselevels or to about these levels are specifically contemplated.

Methods for reducing glomerular hyperfiltration in a subject having orat risk for having diabetes or early stage diabetic nephropathy, themethods comprising administering to the subject a therapeuticallyeffective amount of an agent that inhibits CTGF, are also providedherein, as are methods for reducing glomerular hyperperfusion.

In another aspect, the invention encompasses a method for reducing orpreventing kidney weight gain in a subject having or at risk for havingdiabetes or diabetic nephropathy, the method comprising administering tothe subject a therapeutically effective amount of an agent that inhibitsCTGF.

The invention further provides methods for normalizing glomerularfiltration rate in a subject having or at risk for having diabetes ordiabetic nephropathy, the method comprising administering to the subjecta therapeutically effective amount of an agent that inhibits CTGF. Thediabetic nephropathy can be, for example, early stage, late stage,incipient, or overt diabetic nephropathy. In the case that the diabeticnephropathy is early stage or incipient, the normalization will likelybe a decrease in glomerular filtration rate, while in the case that thediabetic nephropathy is late stage or overt, the normalization willlikely be an increase. Normal GFR in an adult human subject is about 120ml/min. In the event that the subject has a GFR elevated above normallevels, and a decrease in GFR would be desired, methods for decreasingthe GFR to levels below about 150 ml/min., below about 140 ml./min.,below about 130 ml./min, and to about 120 ml/min. are specificallycontemplated. In the event that the subject has GFR impaired ordecreased below normal, methods for increasing the GFR to above about 15ml/min., above about 30 ml/min., above about 60 ml/min., above about 90ml/min., to about 120 ml/min.

In another embodiment, the invention provides a method for reducingglomerular hypertrophy in a subject having or at risk for havingdiabetes or diabetic nephropathy, including early stage, late stage,incipient, or overt diabetic nephropathy, the method comprisingadministering to the subject a therapeutically effective amount of anagent that inhibits CTGF.

Methods for reducing proteinuria in a subject having or at risk forhaving diabetes or diabetic nephropathy are also provided herein, themethod comprising administering to the subject a therapeuticallyeffective amount of an agent that inhibits CTGF. The inventionadditionally encompasses methods for reducing albuminuria in a subjecthaving or at risk for having diabetes or diabetic nephropathy, themethod comprising administering to the subject a therapeuticallyeffective amount of an agent that inhibits CTGF. A method for reducingmicroalbuminuria in a subject having or at risk for having diabetes ordiabetic nephropathy, wherein the diabetic nephropathy is early stage orincipient diabetic nephropathy, the method comprising administering tothe subject a therapeutically effective amount of an agent that inhibitsCTGF, is additionally contemplated, as is a method for reducingmacroalbuminuria in a subject having or at risk for having diabetes ordiabetic nephropathy, wherein the diabetic nephropathy is late stage orovert, the method comprising administering to the subject atherapeutically effective amount of an agent that inhibits CTGF.

Normal urinary albumin excretion levels in adult humans are typicallyabout 15-30 mg per day. Microalbuminuria is typically diagnosed when asubject has a urinary albumin excretion of about 30-300 mg/day.Macroalbuminuria is typically characterized by urinary albumin excretionof greater than about 300 mg/day. The present invention thusspecifically provides methods for decreasing urinary albumin excretionin a subject, the method comprising administering to the subject aneffective amount of an agent that inhibits CTGF, having elevated urinaryalbumin excretion, e.g., urinary albumin excretion elevated above normallevels. Embodiments in which the urinary albumin excretion is reduced tounder about 300 mg/day, under about 200 mg/day, under about 100 mg/day,under about 50 mg/day, and, most preferably, under about 30 mg/day arespecifically contemplated herein.

In certain aspects, the invention provides a method for reducing BUNlevels in a subject having or at risk for having diabetes or diabeticnephropathy, the method comprising administering to the subject atherapeutically effective amount of an agent that inhibits CTGF. NormalBUN levels for adult humans range from 7-20 mg/dL. Therefore, methodsfor reducing BUN levels to below 20 mg/dL. The invention furtherprovides a method for reducing inulin clearance in a subject having orat risk for having diabetes or diabetic nephropathy, the methodcomprising administering to the subject a therapeutically effectiveamount of an agent that inhibits CTGF. In specific aspects, the diabeticnephropathy is late stage diabetic nephropathy or overt diabeticnephropathy.

In yet a further embodiment, the invention provides a method forpreventing, reducing the risk of, or delaying the onset of diabeticcomplications in a subject at risk for developing such complications,the method comprising administering to the subject a therapeuticallyeffective amount of an agent that inhibits CTGF. In various embodiments,the diabetic complications include at least one complication selectedfrom the group consisting of increased creatinine clearance, increasedor decreased glomerular filtration rate, glomerular basement membranethickening, glomerular hyperfiltration, glomerular hyperperfusion,glomerular hypertrophy, increased urinary albumin excretion,microalbumnuria, macroalbuminuria, increased BUN levels, increasedinulin clearance, kidney weight gain, and impaired kidney function.

The invention also encompasses a method for treating incipient diabeticnephropathy in a subject having or at risk for having incipient diabeticnephropathy, the method comprising administering to the subject atherapeutically effective amount of an agent that inhibits CTGF, and amethod for treating early stage diabetic nephropathy in a subject havingor at risk for having early stage diabetic nephropathy, the methodcomprising administering to the subject a therapeutically effectiveamount of an agent that inhibits CTGF. A method for treating overtdiabetic nephropathy in a subject having or at risk for having overtdiabetic nephropathy, the method comprising administering to the subjecta therapeutically effective amount of an agent that inhibits CTGF, isalso contemplated herein.

The present invention contemplates the use of the present methods incombination with other therapies. In one embodiment, the method is usedin combination with another therapy, e.g., to further augmenttherapeutic effect on certain pathological events, etc. The twotreatments may be administered at the same time or consecutively, e.g.,during a treatment time course or following disease progression andremission. In another embodiment, the method is used in combination withanother therapeutic method having a similar or different mode of action,e.g., ACE inhibitors, ARBs, statin, advanced glycation endproduct (AGE)inhibitor, etc. Thus, in a particular embodiment, the present inventionprovides a method for treating diabetic nephropathy in a subject havingor at risk for having diabetic nephropathy, the method comprisingadministering to the subject a therapeutically effective amount of anagent that inhibits CTGF in combination with an inhibiting amount of anangiotensin converting enzyme inhibitor. The present invention furtherprovides a method for treating diabetic nephropathy in a subject havingor at risk for having diabetic nephropathy, the method comprisingadministering to the subject a therapeutically effective amount of anagent that inhibits CTGF in combination with an inhibiting amount of anangiotensin receptor blocker.

Methods for treating progressive renal failure in a subject the methodcomprising administering to the subject a therapeutically effectiveamount of an agent that inhibits CTGF, are provided in one embodiment.In another embodiment, the invention provides a method for reducing therisk or delaying the onset of development of microalbuminuria in asubject, the method comprising administering to the subject atherapeutically effective amount of an agent that inhibits CTGF. In anadditional embodiment, a method for reducing the risk or delaying theonset of development of macroalbuminuria in a subject, the methodcomprising administering to the subject a therapeutically effectiveamount of an agent that inhibits CTGF, is also provided.

In a particular aspect, the invention relates to the present discoverythat CTGF is herein identified as a critical factor in early stageprogressive diseases including diabetic kidney complications andvitreoretinal disorders. Therefore, in one aspect, the invention relatesto a method for treating or preventing early stage aspects of aprogressive disease in a subject having or at risk for having such adisease, the method comprising administering to the subject atherapeutically effective amount of an agent that inhibits CTGF. In afurther aspect, the progessive disease is associated with a growthfactor other than CTGF, and, in a specific aspect, the other growthfactor is VEGF. In one aspect, the progressive disease is a renaldisease, and, in a particular aspect, the progressive disease isassociated with diabetes or with diabetic complications, or is diabeticnephropathy.

The invention additionally encompasses a method for improving kidneyfunction in a subject having or at risk for having impaired kidneyfunction, the method comprising administering to the subject atherapeutically effective amount of an agent that inhibits CTGF.

As summarized in the preceding description, the invention relates to thediscovery that anti-CTGF therapy is effective in treatment or preventionof various physiological features of early stage and late stagy diabeticnephropathy. Accordingly, it is contemplated that the present inventionprovides methods for treating or preventing a renal disorder associatedwith at least one of the features selected from the following: increasedcreatinine clearance; increased glomerular filtration or glomerularhyperfiltration; proteinuria; increased urine albumin excretion;increased glomerular volume; glomerular hypertrophy; increased kidneyweight; glomerular basement membrane thickening; reduced glomerularfiltration rate; increased BUN levels; and increased inulin clearance.In each case, the methods comprise administering to a subject in need ofsuch treatment an effective amount of an agent that inhibits CTGF. Thesemethods specifically cover administration to a subject of the agent thatinhibits CTGF for the express purpose of preventing progression to ordevelopment of any one of the above-described complications.

In preferred embodiments of the above-described methods, the subject isa human subject.

In any of the methods described above, it is particularly contemplatedthat the agent that inhibits CTGF may be a polypeptide, polynucleotide,or small molecule; for example, an antibody that binds to CTGF, anantisense molecule, siRNAs, small molecule chemical compounds, etc. Inparticular, the present invention contemplates that inhibiting CTGF canbe accomplished by any of the means well-known in the art for modulatingthe expression and activity of CTGF. Use of anti-CTGF agent, forexample, a human monoclonal antibody directed against CTGF, ispreferred, although any method of inhibiting expression of the geneencoding CTGF, inhibiting production of CTGF, or inhibiting activity ofCTGF is contemplated by the present invention. For example, smallmolecule compounds may be used to inhibit CTGF expression, production,or activity. As CTGF expression is inhibited by cyclic nucleotide, sucha compound may include, e.g., a cyclic nucleotide analog or aphospodiesterase (PDE) inhibitor. (See, e.g., Duncan et al. (1999) FASEBJ. 13:1774-1786.) Further, polynucleotides including small interferingribonucleic acids (siRNAs), micro-RNAs (mRNAs), ribozymes, andanti-sense sequences may be used in the present methods to inhibitexpression and/or production of CTGF. (See, e.g., Kondo et al. (2000)Biochem Biophys Res Commun 278:119-124.) Such techniques are well-knownto those of skill in the relevant art. Exemplary antibodies for use inthe methods of the present invention are described, e.g., inInternational Publication No. WO 2004/108764, which is incorporatedherein by reference in its entirety.

DESCRIPTION OF TH INVENTION

It is to be understood that the invention is not limited to theparticular methodologies, protocols, cell lines, assays, and reagentsdescribed herein, as these may vary. It is also to be understood thatthe terminology used herein is intended to describe particularembodiments of the present invention, and is in no way intended to limitthe scope of the present invention as set forth in the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural references unlesscontext clearly dictates otherwise. Thus, for example, a reference to “afragment” includes a plurality of such fragments, a reference to an“antibody” is a reference to one or more antibodies and to equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are now described. All publications cited hereinare incorporated herein by reference in their entirety for the purposeof describing and disclosing the methodologies, reagents, and toolsreported in the publications that might be used in connection with theinvention. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, molecularbiology, cell biology, genetics, immunology and pharmacology, within theskill of the art. Such techniques are explained fully in the literature.See, e.g., Gennaro, A. R., ed. (1990) Remington's PharmaceuticalSciences, 18th ed., Mack Publishing Co.; Colowick, S. et al., eds.,Methods In Enzymology, Academic Press, Inc.; Handbook of ExperimentalImmunology, Vols. I-IV (D. M. Weir and C. C. Blackwell, eds., 1986,Blackwell Scientific Publications); Maniatis, T. et al., eds. (1989)Molecular Cloning: A Laboratory Manual, 2nd edition, Vols. I-III, ColdSpring Harbor Laboratory Press; Ausubel, F. M. et al., eds. (1999) ShortProtocols in Molecular Biology, 4th edition, John Wiley & Sons; Ream etal., eds. (1998) Molecular Biology Techniques: An Intensive LaboratoryCourse, Academic Press); PCR (Introduction to Biotechniques Series), 2nded. (Newton & Graham eds., 1997, Springer Verlag).

The invention relates in part to the discovery that connective tissuegrowth factor (CTGF) plays a key role in specific early stage aspects ofrenal disease including, e.g., glomerular hyperfiltration, increasedglomerular permeability, increased glomerular filtration rate,microalbuminuria, etc. CTGF had previously been associated with specificlate stage aspects of kidney disease, e.g., glomerulosclerosis andtubulointerstitial fibrosis, but had not been identified as a criticaltarget for affecting various features of early stage renal pathologies.Methods for treating or preventing renal disorders including, e.g.,diabetic nephropathy, and methods for treating or preventing associatedpathologies are specifically contemplated.

The present invention provides methods and compositions for reducing orameliorating in a subject complications associated with multiple,distinct pathological processes associated with renal disorders, e.g.,diabetic nephropathy, by inhibiting CTGF. In some embodiments, thesubject is an animal, more preferably a mammal, and most preferably ahuman.

The present invention also provides compositions for use in the methodsdescribed herein. Such compositions may include small moleculecompounds; peptides and proteins including antibodies or functionallyactive fragments thereof; and polynucleotides including smallinterfering ribonucleic acids (siRNAs), micro-RNAs (mRNAs), ribozymes,and anti-sense sequences. (See, e.g., Zeng (2003) Proc Natl Acad Sci USA100:9779-9784; and Kurreck (2003) Eur J. Biochem 270:1628-1644.)

The present invention is based in part on the discovery of unexpectedbenefits of inhibition of CTGF in treatment of multiple and specificaspects of renal disorders, e.g., diabetic nephropathy. The presentinvention provides data demonstrating that inhibition of CTGF reducedvarious pathological aspects of renal disease not previously associatedwith CTGF. In certain aspects, the present invention provides evidencethat inhibition of CTGF provides a therapeutic approach to treat orprevent specific physiological aspects of diabetic nephropathypreviously associated with biological and pathological activities ofVEGF, such as, for example, glomerular hyperfiltration andhyperperfusion.

Diabetic Nephropathy

Diabetes is a major cause of morbidity and mortality worldwide, withapproximately 40% of all individuals with diabetes developing diabeticnephropathy, requiring either kidney dialysis or transplantation.Diabetes is the leading cause of end stage renal disease, and therefore,any individual diagnosed with diabetes is at risk for the development ofdiabetic nephropathy.

Progression of diabetic nephropathy is characterized by a fairlypredictable pattern of events. Generally, the time course of developmentof diabetic nephropathy is as follows. Glomerular hyperfiltration andrenal hypertrophy occur in the first years after the onset of diabetesand are reflected by increased glomerular filtration rate (e.g., from anormal glomerular filtration rate of about 120 ml/min to about 150ml/min in humans). During the first 5 years of diabetes, pathologicalchanges, such as glomerular hypertrophy, thickening of the glomerularbasement membrane, and glomerular mesangial volume expansion, areobserved. Glomerular filtration rate gradually returns to normal. After5 to 10 years of diabetes, individuals begin to excrete small amounts ofalbumin in the urine (microalbuminuria). Microalbuminuria (diabeticindividuals with microalbuminuria are referred to as having incipientdiabetic nephropathy) is an important predictor of progression to overtdiabetic nephropathy (characterized, in part, by macroalbuminuria orovert proteinuria). The basement membrane thickening and glomerularvolume expansion seen in early stages of the disease can accumulate inlate stage diabetic nephropathy, leading to obliteration of thecapillary lumen, and, eventually, to glomerulosclerosis. Once overtdiabetic nephropathy is present, a steady decline in the glomerularfiltration rate occurs, and approximately half of individuals reachend-stage renal disease in 7 to 10 years.

Clinically, the stages of development and progression of diabeticnephropathy in humans have been well described. Stage I diabeticnephropathy is associated with increased kidney (i.e., glormerular)filtration (i.e., hyperfiltration, resulting from increased blood flowthrough the kidneys and glomeruli), increased glomerular filtrationrate, glomerular hypertrophy, and enlarged kidneys. Stage II diabeticnephropathy is a clinically silent phase associated with continuedhyperfiltration and kidney hypertrophy. Thickening of the glomerularbasement membrane and mesangial expansion occurs. Stage III diabeticnephropathy (also known as incipient diabetic nephropathy) is associatedwith microalbuminuria and micro proteinuria. Microalbuminuria is definedas 30 to 300 mg/day urinary albumin in a 24-hour collection, 20-200μg/min urinary albumin, or 30 to 300 μg/mg creatinine in a spotcollection. The kidneys progressively lose the ability to filter wasteand blood levels of creatinine and urea-nitrogen increase. Glomerularbasement membrane thickening and mesangial expansion continue to occurwith increasing severity. Stage IV diabetic nephropathy (also known asovert diabetic nephropathy) is associated with macroalbuminuria (i.e.,clinical albuminuria) and creatinine and blood urea-nitrogen (BUN)levels in the blood continue to rise. Macroalbuminuria is defined asgreater than 300 mg/day urinary albumin in a 24-hour collection, greaterthan 200 μg/min urinary albumin, or greater than 300 μg/mg creatininespot collection. Once overt diabetic nephropathy occurs, glomerularfiltration rate gradually falls over a period of several years. Stage Vdiabetic nephropathy occurs with end-stage renal disease and kidneyfailure.

Hyperfiltration and Hyperperfusion

Early stage diabetic nephropathy is associated with impaired renalfunction, characterized in part by glomerular hyperfiltration andhyperperfusion. Glomerular hyperfiltration is a glomerular adaptation tonephron loss associated with hyperglycemia and diabetes. With loss offunctioning nephron mass, the remaining functional nephrons hypertrophyand take on an increased workload, thereby attempting to minimize theoverall loss of renal function. As a result, glomerular hyperfiltrationand hyperperfusion occur.

Glomerular hyperfiltration and hyperperfusion are reflected as increasedglomerular filtration rate. Glomerular filtration rate is a measurementof the volume of filtrate made by the kidneys per minute. Measurement ofglomerular filtration rate in human subjects has been accepted as thebest overall index of kidney function in health and disease. (Smith,Diseases of the kidney and urinary tract, In: Structure and Function inHealth and Disease, New York; Oxford Univ. Press, 1951:836-887.)Glomerular filtration rate can be determined by various methods, such asby measuring the urinary clearance of a filtration marker, such asinulin, iothalamate, or iohexol. More commonly, glomerular filtrationrate is estimated by determining clearance of creatinine, a proteinproduced by muscle and released into the blood. Creatinine clearance(often expressed as ml/min) can be determined by comparing the level ofcreatinine collected in urine over a given period of time, e.g., 12 or24 hours, with the creatinine level in blood. A typical creatinineclearance rate is about 97 to 137 ml/min in adult males, and about 88 to128 ml/min in adult females.

In clinical practice, creatinine clearance is most often estimated fromthe serum creatinine concentration. Creatinine clearance is relateddirectly to the urine creatinine excretion and inversely to serumcreatinine concentration. Various formulas that provide estimates ofcreatinine clearance, and therefore estimates of glomerular filtrationrate, using parameters such as serum creatinine concentration, age, sex,and body size, have been developed and are standard in the art. (See,e.g., Cockcroft and Gault (1976) Nephron 16:3141; Levey et al (1999)Annals of Internal Medicine 130:462-470; Rule et al (2004) Ann InternMed 141:929-937.)

Methods and compounds of the present invention reduced creatinineclearance in an animal model of diabetes. (See, e.g., Example 1.)Therefore, the present invention provides methods and compounds forreducing creatinine clearance in a subject with increased or elevatedcreatinine clearance or in which creatinine clearance is elevated abovenormal levels. The present invention demonstrates that inhibition ofCTGF (e.g., by administration of an antibody to CTGF) reduces creatinineclearance associated with nephropathy, and in particular, diabeticnephropathy. Increased creatinine clearance is associated withglomerular hyperfiltration, hyperperfusion, hypertrophy, and increasedglomerular filtration rate, and is indicative of altered or impairedrenal function in early stages of developing nephropathy, e.g., diabeticnephropathy. In one aspect, the present invention provides methods andcompounds for reducing creatinine clearance by inhibiting CTGF. Inanother aspect, the present invention provides methods and compounds forreducing glomerular creatinine permeability and restoring glomerularselectivity and function by inhibiting CTGF. In another aspect, methodsand compounds are provided for treating or preventing glomerularhypertrophy, hyperfiltration, and hyperperfusion associated withhyperglycemia or diabetes by inhibiting CTGF. In yet another aspect,methods and compounds are provided for treating or preventing glomerularhypertrophy, hyperfiltration, and hyperperfusion associated with renaldiseases, and, in particular, diabetic nephropathy, by inhibiting CTGF.In one aspect, the renal disease is early stage diabetic nephropathy.

In other aspects, the present invention provides methods and compoundsfor reducing glomerular filtration rate in a subject with an increasedglomerular filtration rate by inhibiting CTGF. In one aspect, thepresent invention provides methods and compounds for reducing glomerularfiltration rate by administering to a subject having or at risk forhaving an impaired or increased glomerular filtration rate an agent thatinhibits CTGF. In one aspect, the impaired glomerular filtration andincreased glomerular filtration rate are associated with early stagekidney disease.

In certain embodiments, the present invention provides methods andcompounds for treating a renal disorder associated with or characterizedby increased creatinine clearance by administering to a subject havingor at risk for having the disorder an agent that inhibits CTGF, thustreating or preventing the disorder. In other embodiments, the presentinvention provides methods and compounds for treating a renal disorderassociated with or characterized by increased glomerular filtration orglomerular hyperfiltration by administering to a subject having or atrisk for having the disorder an agent that inhibits CTGF, thus treatingor preventing the disorder.

Methods and compounds of the present invention were found to increasethe glomerular filtration rate in an animal model of late stage diabeticnephropathy. (See Example 3.) Therefore, the present invention providesmethods and compounds for increasing or normalizing glomerularfiltration rate in a subject with a reduced or impaired glomerularfiltration rate or in which the glomerular filtration rate is belownormal by inhibiting CTGF. In one aspect, the present invention providesmethods and compounds for increasing or normalizing the glomerularfiltration rate by administering to a subject having or at risk forhaving an impaired or reduced glomerular filtration rate an agent thatinhibits CTGF. In another aspect, the impaired glomerular filtrationrate and reduced glomerular filtration rate are associated with latestage kidney disease or overt diabetic nephropathy.

In one aspect, the present invention provides methods and compounds fortreating or preventing a renal disorder associated with impairedglomerular filtration rate and reduced glomerular filtration rate byadministering to a subject having or at risk for having the disorder anagent that inhibits CTGF, thus treating or preventing the disorder. Inanother aspect, the impaired glomerular filtration rate and reducedglomerular filtration rate are associated with late stage kidneydisease.

It is contemplated that the present methods can be applied to improvingrenal function, normalizing glomerular filtration rate, reducingglomerular hyperfiltration and hyperperfusion, or reducing creatinineclearance in a subject with any clinically accepted standard ofmeasurement indicative of nephropathy or renal disease, or a subject atrisk for developing such a renal disorder. In certain embodiments, thesubject has diabetic kidney disease. In various embodiments, the subjecthas stage I kidney disease, stage II kidney disease, stage III kidneydisease, stage IV kidney disease, or stage V kidney disease.

The present methods are applied to preventing, reducing, or delaying theonset of renal complications associated with early stage kidney diseasein a subject at risk for developing such complications, or tomanufacture of a medicament for a subject, preferably a human subject,having any of the disorders and features associated with early stagekidney disease discussed herein. In one aspect, the subject hasdiabetes. Diabetes can be determined by any measure accepted andutilized by those of skill in the art. A human subject would bediagnosed with diabetes with a blood glucose level above about 200 mg/dL(as determined in a fasting blood glucose test, an oral glucosetolerance test, or a random blood glucose test). Therefore, in certainaspects, it is contemplated that a human subject having a blood glucoselevel above about 200 mg/dL is a suitable subject for treatment with themethods or use of medicaments provided by the present invention.

Other suitable subjects contemplated for treatment with the presentmethods have impaired glomerular filtration rate. In one embodiment, thehuman subject has a glomerular filtration rate above normal glomerularfiltration rate, e.g., above about 120 ml/min. Therefore, it iscontemplated that a human subject having a glomerular filtration rateabove about 120 ml/min, above about 130 ml/min, above about 140 ml/min,or above about 150 ml/min is a suitable subject for treatment with themethods or use of medicaments provided by the present invention. It isfurther contemplated, in various embodiments, that the methods forreducing glomerular filtration rate in a subject with increasedglomerular filtration rate (e.g., in a subject with glomerularhyperfiltration and hyperperfusion) can be applied to reducingglomerular filtration rate in a human subject to a level below about 150ml/min, below about 140 ml/min, below about 130 ml/min, or to a level ofabout 120 ml/min.

Methods and compounds of the present invention reduced the increase inkidney weight associated with diabetes and early stage diabeticnephropathy in an animal model of diabetes. (See Example 1.) Therefore,a method for treating or preventing a renal disorder associated withincreased kidney weight, the method comprising administering to asubject having or at risk for having the disorder an agent that inhibitsCTGF, thus treating or preventing the disorder, is contemplated by thepresent invention. The invention further contemplates a method fortreating or preventing a renal disorder associated with increasedglomerular volume, the method comprising administering to a subjecthaving or suspected of having the disorder an agent that inhibits CTGF,thus treating or preventing the disorder.

The present methods and compounds are also applied to preventing,reducing, or delaying the onset of renal complications associated withlate stage kidney disease in a subject at risk for developing suchcomplications, or manufacture of a medicament for a subject, preferablya human subject, having any of the disorder and conditions associatedwith late stage kidney disease discussed herein. In one aspect, thehuman subject has a glomerular filtration rate below a normal glomerularfiltration rate, e.g., below about 120 ml/min. Therefore, it iscontemplated that a human subject having a glomerular filtration ratebelow about 120 ml/min, below about 90 ml/min, below about 60 ml/min,below about 30 ml/min, or below about 15 ml/min is a suitable subjectfor treatment with the methods or use of medicaments provided by thepresent invention.

It is further contemplated, in various embodiments, that the methods forincreasing glomerular filtration rate in a human subject with reduced orimpaired glomerular filtration rate (e.g., in a subject with overtdiabetic nephropathy) can be applied to increase glomerular filtrationrate to a level above about 15 ml/min, above about 30 ml/min, aboveabout 60 ml/min, above about 90 ml/min, and to a level of about 120m/min.

In certain embodiments, the renal disorder is associated with Type 1 orType 2 diabetes. In other embodiments, the renal disorder is diabeticnephropathy.

Microalbuminuria

Early clinical evidence of nephropathy, including diabetic nephropathy,is the appearance of low but abnormal levels of albumin in the urine, acondition referred to as microalbunimuria. Individuals withmicroalbuminuria are referred to as having incipient nephropathy, or, ifassociated with diabetes, incipient diabetic nephropathy. Diabeticindividuals with microalbuminuria have a 42% increased risk ofprogression to overt diabetic nephropathy compared to those withnormoalbuminuria (Bruno et al, 2003, Diabetes Care 26:2150-2155).Therefore, the appearance and development of microalbuminuria inindividuals with diabetes is associated with a greatly-increased risk ofprogression to overt diabetic nephropathy (i.e., macroalbuminuria) andeventual end-stage renal disease and kidney failure. (See, e.g.,Mogensen and Christensen (1984) N Engl J. Med 311:89-93; Mogensen et al(1983) Diabetes 32[Suppl 2]:64-78; Viberti et al (1982) Lancet1:1430-1432.)

Microalbuminuria can be determined by various methods, including: (1)measurement of the albumin-to-creatinine ratio in a random spot urinecollection; (2) 24-hour urine collection with creatinine, allowing thesimultaneous measurement of creatinine clearance; and (3) timed (e.g.,4-hour or overnight) collection. Normal urinary albumin excretion inhumans is less than 30 μg/mg creatinine (spot collection), less than 30mg/24-hours (24-hour collection), or less than 20 μg/min (timedcollection). Microalbuminuria in humans is having urinary albuminexcretion of 30 to 299 μg/mg creatinine (spot collection), 30 to 299mg/24-hours (24-hour collection), or 20 to 199 μg/min (timedcollection). Macroalbuminuria (e.g., clinical albuminuria) in humans ishaving urinary albumin excretion of greater than or equal to 300 μg/mgcreatinine (spot collection), greater than or equal to 300 mg/24-hours(24-hour collection), or greater than or equal to 200 μg/min (timedcollection).

The present invention demonstrates for the first time that inhibition ofCTGF (e.g., by administration of an antibody to CTGF) reduces urinaryalbumin excretion associated with nephropathy, and in particular,diabetic nephropathy. (See, e.g., Example 1.) Increased urinary albuminexcretion is associated with changes in glomerular albumin permeabilityand selectivity, and is indicative of altered or impaired renal functionin early stages of developing nephropathy. In one aspect, the presentinvention provides methods for reducing urinary albumin excretion byinhibiting CTGF. In another aspect, the present invention providesmethods for reducing glomerular albumin permeability and restoringglomerular selectivity by inhibiting CTGF. In yet another aspect, thepresent invention provides methods for reducing microalbuminuria byinhibiting CTGF. By reducing microalbuminuria and urinary albuminexcretion, the present methods, therefore, provide a means for treatingearly stage kidney disease and incipient nephropathy.

As described above, in early stage kidney disease, the onset anddevelopment of microalbuminuria (i.e., incipient nephropathy) isassociated with an increased risk of development of macroalbuminuia,overt nephropathy, end stage renal disease, and kidney failure inindividuals with diabetes. Methods and compositions of the presentinvention, therefore, are also applied to preventing, reducing, ordelaying the onset of or reduce the risk of developing renalcomplications associated with late stage kidney disease, includingmacroalbuminuia, overt nephropathy, end stage renal disease, and kidneyfailure, in a subject at risk for developing such complications.

The present invention demonstrates that inhibition of CTGF (e.g., byadministration of an antibody to CTGF) reduces proteinuria, BUN levels,and creatinine clearance associated with nephropathy. Increasedproteinuria, BUN levels, and creatinine clearance are indicative ofaltered or impaired renal function and development of nephropathy. Inone aspect, the present invention provides methods and compounds forreducing proteinuria by inhibiting CTGF. In anther aspect, the presentinvention provides methods and compounds for reducing BUN levels byinhibiting CTGF. In another aspect, methods and compounds are providedfor reducing creatinine clearance by inhibiting CTGF.

The present invention demonstrates that inhibition of CTGF (e.g., byadministration of an antibody to CTGF) improves kidney function. Asdiabetic nephropathy progresses to late stage kidney disease, glomerularfiltration rate decline, as measured, for example, by decreased inulinclearance, is indicative of altered or impaired kidney function. Thepresent invention further demonstrates that inhibition of CTGF (e.g., byadministration of an antibody to CTGF) improved the impaired or reducedglomerular filtration rate associated with late stage kidney disease.(See Example 3.) In one aspect, the present invention provides methodsand compounds for increasing glomerular filtration rate by inhibitingCTGF. In another aspect, the present invention provides methods andcompounds for decreasing inulin clearance by inhibiting CTGF. In yetanother aspect, methods and compounds are provided for treating orpreventing impaired kidney function, in particular impaired kidneyfunction associated with nephropathy, such as diabetic nephropathy, byinhibiting CTGF. In other aspects, the nephropathy is associated withdecreased glomerular filtration rate, macroalbuminuria, or overtnephropathy.

Late stage diabetic nephropathy is associated with various pathologicaland morphological changes in the kidney. Such changes include mesangialexpansion, associated with increased matrix production and accumulationof mesangial extracellular matrix; mesangial cell expansion; glomerularbasement membrane thickening, which in late stage diabetic nephropathyis associated with glomerulosclerosis; and development oftubulointerstitial fibrosis. (Gilbert et al (1999) Kidney Int56:1627-1673.) Glomerulosclerosis and tubulointerstitial fibrosis arethe structural late stage kidney disease hallmarks of advanced diabeticnephropathy with renal insufficiency, resulting in reduction inglomerular filtration rate and, possibly, end stage renal disease andkidney failure.

Prior to the present invention, CTGF had been associated with featuresof late stages of renal pathology, specifically, production of excessextracellular matrix, excess mesangial matrix expansion, and developmentof glomeruloscleorsis and tubulointerstitial fibrosis. (See, e.g.,International Publication No. WO 00/13706.) It was thought that otherfactors, e.g., VEGF, were responsible for processes, pathologies andvarious features associated with early stage renal disease, e.g.,hyperfiltration and increased glomerular permeability. In contrast, thepresent invention provides data demonstrating that it is CTGF that playsa key role in the development and progression of early stage as well aslate stage aspects of nephropathy, and thus represents an ideal targetfor a complete and effective therapeutic approach to diabeticnephropathy.

The present invention provides methods for treating and preventingvarious clinical and pathological aspects of early stage as well as latestage diabetic nephropathy. Specifically, methods and compositions ofthe present invention are useful for treating or preventing glomerularhyperfiltration and mesangial matrix expansion. Therefore, the presentinvention contemplates methods for treating various aspects of renaldisease, including features of early stage diabetic kidney disease, suchas, e.g., renal and glomerular hypertrophy and hyperfiltration (measuredas increased creatinine clearance, increased urinary albumin excretion,increased glomerular filtration rate, etc.), and features of late stagediabetic kidney disease (decreased glomerular filtration rate, mesangialmatrix expansion, basement membrane thickening, etc.).

The present invention provides methods, and compositions for usetherein, for treating a disorder or condition wherein connective tissuegrowth factor (CTGF) is a mediating factor. Several CTGF-associateddisorders have been described in the literature; however, until thepresent invention, CTGF was primarily associated with fibroproliferativeconditions, particularly those associated with TGFβ. Although numerousdisorders involve fibroproliferative processes, and the treatment ofthese disorders using therapeutics directed at providing or preventingCTGF activity have been suggested, the present invention extends theunderstanding and, thus, the use of CTGF-directed therapies to treatmentof various non-fibroproliferative conditions and complicationsassociated with diabetic nephropathy and renal disorders.

The methods of the present invention, e.g., inhibiting CTGF, effectivelyreduce hyperfiltration by the kidney and normalize or restore kidneyfunction as measured, e.g., by glomerular filtration rate, urinaryalbumin excretion, albuminuria, and/or proteinuria. Thus, the methodsand compositions of the present invention can be used to treat patientsat risk for diabetic nephropathy, including, for example, early stagediabetic nephropathy and incipient diabetic nephropathy. Such subjectsinclude individuals diagnosed with hyperglycemia, hypertension, and/ordiabetes. Additionally, the methods of the present invention can be usedto treat patients diagnosed with a kidney disorder such asglomeruloscerosis, glomerulamephritis, or diabetic nephropathy.

The methods of the present invention, e.g., inhibiting CTGF, reducemesangial matrix expansion and glomerular basement membrane thickening.Thus, the methods of the present invention can be used to treat patientsat risk for diabetic nephropathy to prevent albuminuria, reducedglomerular filtration rate, and the like. Such subjects includeindividuals diagnosed with hyperglycemia, hypertension, and/or diabetes.Additionally, the methods of the present invention can be used to treatpatients having overt diabetic nephropathy or another renal disordersuch as glomeruloscerosis, glomerularnephritis, etc.

Therefore, in one aspect, the present invention contemplates methods oftreating or preventing processes associated with early stage renaldisease or late stage renal disease by inhibiting CTGF. Thesepathological conditions include, for example, hyperfiltration,albuminuria, proteinuria, glomerular hypertrophy, and mesangial volumeexpansion. Use of the present methods to treat or prevent aspects ofearly stage and of late stage renal disease previously associated withVEGF and TGFβ is specifically contemplated. As stated above, the methodscan be used to treat patients at risk for diabetic nephropathy or anassociated pathology, and to treat patients having a renal disorder suchas glomeruloscerosis, glomerulonephritis, diabetic nephropathy, etc.

The present invention contemplates the use of the present methods incombination with other therapies. In one embodiment, the method is usedin combination with another therapy, e.g., to further augmenttherapeutic effect on certain pathological events, etc. The twotreatments may be administered at the same time or consecutively, e.g.,during a treatment time course or following disease progression andremission. In another embodiment, the method is used in combination withanother therapeutic method having a similar or different mode of action,e.g., an ACE inhibitor, ARBs, statin, advanced glycation endproduct(AGE) inhibitor, etc. Current therapeutic approaches to treat diabeticnephropathy are known by one of skill in the art, and include, forexample, ACE inhibitors, angiotensin receptor blockers, statins,advanced glycation endproduct inhibitors, hepatocyte growth factor genetherapy, pyridoxamine, Enapril, PPAR antagonists, sulfonylureas, matrixmetalloproteinase inhibitors, COX-2 inhibitors, pirfenidone, sulodexide,high-dose thiamine and Benfotiamine, calcium channel blockers, etc. Useof any of these therapeutic agents in combination with the use ofmethods of the present invention is specifically contemplated.

The present invention represents the first time therapeutic efficacy oftwo distinct pathological aspects associated with renal disease (forexample, early stage features and late stage features of diabeticnephropathy) has been demonstrated. Although anti-CTGF therapy isexemplified herein using a human monoclonal antibody directed againstCTGF, any method of inhibiting expression of the gene encoding CTGF,inhibiting production of CTGF, or inhibiting activity of CTGF iscontemplated by the present invention. For example, small moleculecompounds may be used to inhibit CTGF expression, production, oractivity. As CTGF expression is inhibited by cyclic nucleotide, such acompound may include, e.g., a cyclic nucleotide analog or aphospodiesterase (PDE) inhibitor. (See, e.g., Duncan et al. (1999) FASEBJ. 13:1774-1786.) Further, polynucleotides including small interferingribonucleic acids (siRNAs), micro-RNAs (mRNAs), ribozymes, andanti-sense sequences may be used in the present methods to inhibitexpression and/or production of CTGF. (See, e.g., Kondo et al. (2000)Biochem Biophys Res Commun 278:119-124; and Shimo et al., supra.) Suchtechniques are well-known to those of skill in the relevant art.

The present invention provides exemplary evidence that the methodsdescribed herein, using an anti-CTGF monoclonal antibody in animalmodels of diabetes, provide improvement in creatinine clearance andglomerular hypertrophy, and a decrease in kidney weight, an indicator ofTGFβ-induced and CTGF-mediated glomerular fibrosis and mesangialexpansion. Thus, the methods of the present invention ameliorate twopathologies contributing to diabetic nephropathy, i.e., mesangialexpansion and glomerular filtration.

In certain aspects, the present invention provides methods andcompositions for treating a disorder associated with TGFβ by inhibitingCTGF. In other aspects, the present invention provides methods andcompositions for treating a disorder associated with VEGF by inhibitingCTGF. In yet other aspects, the present invention provides methods andcompositions for treating a disorder associated with TGFβ and VEGF byinhibiting CTGF. It is further contemplated in the present inventionmethods and compositions for treating a disorder associated with othergrowth factors, e.g., IGF-1, endothelin, etc.

In one aspect, the present invention provides a method for treating orpreventing a renal disorder associated with increased creatinineclearance, the method comprising administering to a subject having or atrisk for having the disorder an agent that inhibits CTGF (e.g., inhibitsor reduces CTGF expression or CTGF activity), thus treating orpreventing the renal disorder. In another aspect, the present inventionprovides methods for treating or preventing a renal disorder associatedwith increased glomerular filtration and hyperfiltration byadministering to a subject having or at risk for having the disorder anagent that inhibits CTGF, thus treating or preventing the disorder. Inanother aspect, the present invention provides methods for treating orpreventing a renal disorder associated with basement membrane thickeningby administering to a subject having or at risk for having the disorderan agent that inhibits CTGF, thus treating or preventing the disorder.In another aspect, the present invention provides methods for treatingor preventing a renal disorder associated with increased urine volume byadministering to a subject having or at risk for having the disorder anagent that inhibits CTGF, thus treating or preventing the disorder. Amethod for treating a renal disorder associated with increased urinaryalbumin excretion by administering to a subject having the disorder orat risk for having the disorder an agent that inhibits CTGF, thustreating or preventing the renal disorder, is also provided.

In one aspect, the present invention provides methods for reducingcreatinine clearance in a subject in need of such treatment, the methodcomprising administering to the subject an agent that inhibits CTGF.Methods for reducing urinary albumin excretion, reducing glomerularfiltration and hyperfiltration, reducing glomerular volume expansion, orreducing kidney weight increase in a subject in need of such treatmentare also provided, the methods comprising administering to the subjectan agent that inhibits CTGF. In one embodiment, the present inventionprovides a method for treating or preventing proteinuria associated withrenal disease, the method comprising administering to a subject havingor at risk for having the renal disease an agent that inhibits CTGF. Ina further embodiment, the proteinuria is albuminuria. In respectiveembodiments, the albuminuria is microalbumnuria or macroalbuminuria. Inanother embodiment, the present invention provides a method for treatingor preventing basement membrane thickening in the kidney, the methodcomprising administering to a subject having or as risk for havingbasement membrane thickening in the kidney an agent that inhibits CTGF.In yet another embodiment, the present invention provides a method forreducing or preventing increased urine volume by administering to asubject having or at risk for having increased urine volume an agentthat inhibits CTGF.

Methods of the present invention include administering to a subject inneed a therapeutically effective amount of an agent that inhibits CTGF(e.g., reduces CTGF expression or activity). In certain embodiments, theagent is an antibody to CTGF. In a preferred embodiment, the antibody isa monoclonal antibody to CTGF. In another preferred embodiment, theantibody is a human or humanized antibody to CTGF. In anotherembodiment, the agent is a small molecule. In another embodiment, theagent is an antisense oligonucleotide.

Various agents that inhibit CTGF have been identified. Antibodies thatbind to CTGF are described in U.S. Pat. No. 5,408,040; InternationalPublication No. WO 99/07407; International Publication No. WO 99/33878;and International Publication No. WO 00/35936. An exemplary antibody furuse in the methods of the present invention has been described inInternational Publication No. WO 2004/108764, incorporated by referenceherein in its entirety. Such antibodies, or fragments thereof, can beadministered by various means known to those skilled in the art. Forexample, antibodies are often injected intravenously, intraperitoneally,or subcutaneously.

Small molecule inhibitors of CTGF expression and/or activity have alsobeen described; for example, International Publication No. WO 96/38172identifies modulators of cAMP such as cholera toxin and 8Br-cAMP asinhibitors of CTGF expression. Therefore, compounds identified as, e.g.,prostaglandin and/or prostacyclin analogs such as Iloprost (see, e.g.,International Publication No. WO 00/02450; Ricupero et al. (1999) Am J.Physiol 277:L1165-1171; also, see Ertl et al. (1992) Am Rev Respir Dis145:A19), and potentially phosphodiesterase IV inhibitors (see, e.g.,Kohyama et al. (2002) Am J. Respir Cell Mol Biol 26:694-701), may beused to modulate CTGF expression. Also, inhibitors of serine/threoninemitogen activated protein kinases, particularly p38, cyclin-dependentkinase, e.g. CDK2, and glycogen synthase kinase (GSK)-3 have also beenimplicated in decreased CTGF expression. (See, e.g., Matsuoka et al.(2002) Am J. Physiol Lung Cell Mol Physiol 283:L103-L112; Yosimichi etal. (2001) Eur J. Biochem 268:6058-6065; International Publication No.WO 01/38532; and International Publication No. WO 03/092584.) Suchagents can be used to reduce expression of CTGF and thereby ameliorateor prevent the pathological processes induced by CTGF in jointdisorders. Such compounds can be formulated and administered accordingto established procedures within the art.

Antisense technologies, including small interfering ribonucleic acids(siRNAs), micro-RNAs (mRNAs), ribozymes, and anti-sense sequencesdirected to CTGF expression may also be used to treat joint disorders.(See, e.g., Zeng (2003) Proc Natl Acad Sci USA 100:9779-9784; andKurreck (2003) Eur J. Biochem 270:1628-1644.) Antisense constructs thattarget CTGF expression have been described and utilized to reduce CTGFexpression in various cell types. (See, e.g., International PublicationNo. WO 96/38172; International Publication No. WO 00/27868;International Publication No. WO 00/35936; International Publication No.WO 03/053340; Kothapalli et al. (1997) Cell Growth Differ 8(1):61-68;Shimo et al. (1998) J. Biochem (Tokyo) 124(1):130-140; and Uchio et al.p2004) Wound Repair Regen 12:60-66.) Such antisense constructs can beused to reduce expression of CTGF and thereby ameliorate or prevent thepathological processes induced by CTGF in joint disorders. Suchconstructs can be designed using appropriate vectors and expressionalregulators for cell- or tissue-specific expression and constitutive orinducible expression. Such genetic constructs can be formulated andadministered according to established procedures within the art.

Pharmaceutical Formulations and Routes of Administration

The compositions of the present invention can be delivered directly orin pharmaceutical compositions containing excipients, as is well knownin the art. Present methods of treatment can comprise administration ofan effective amount of a compound of the present invention to a subjecthaving or at risk for diabetic nephropathy; particularly a disorderassociated with, for example, glomerular hyperfiltration andhyperperfusion, microalbuminuria, incipient diabetic nephropathy,macroalbuminuria, overt nephropathy, etc. In a preferred embodiment, thesubject is a mammalian subject, and in a most preferred embodiment, thesubject is a human subject.

An effective amount, e.g., dose, of compound or drug can readily bedetermined by routine experimentation, as can an effective andconvenient route of administration and an appropriate formulation.Various formulations and drug delivery systems are available in the art.(See, e.g., Gennaro, ed. (2000) Remington's Pharmaceutical Sciences,supra; and Hardman, Limbird, and Gilman, eds. (2001) The PharmacologicalBasis of Therapeutics, supra.)

Suitable routes of administration may, for example, include oral,rectal, topical, nasal, pulmonary, ocular, intestinal, and parenteraladministration. Primary routes for parenteral administration includeintravenous, intramuscular, and subcutaneous administration. Secondaryroutes of administration include intraperitoneal, intra-arterial,intra-articular, intracardiac, intracistemal, intradermal,intralesional, intraocular, intrapleural, intrathecal, intrauterine, andintraventricular administration. The indication to be treated, alongwith the physical, chemical, and biological properties of the drug,dictate the type of formulation and the route of administration to beused, as well as whether local or systemic delivery would be preferred.

Pharmaceutical dosage forms of a compound of the invention may beprovided in an instant release, controlled release, sustained release,or target drug-delivery system. Commonly used dosage forms include, forexample, solutions and suspensions, (micro-) emulsions, ointments, gelsand patches, liposomes, tablets, dragees, soft or hard shell capsules,suppositories, ovules, implants; amorphous or crystalline powders,aerosols, and lyophilized formulations. Depending on route ofadministration used, special devices may be required for application oradministration of the drug, such as, for example, syringes and needles,inhalers, pumps, injection pens, applicators, or special flasks.Pharmaceutical dosage forms are often composed of the drug, anexcipient(s), and a container/closure system. One or multipleexcipients, also referred to as inactive ingredients, can be added to acompound of the invention to improve or facilitate manufacturing,stability, administration, and safety of the drug, and can provide ameans to achieve a desired drug release profile. Therefore, the type ofexcipient(s) to be added to the drug can depend on various factors, suchas, for example, the physical and chemical properties of the drug, theroute of administration, and the manufacturing procedure.Pharmaceutically acceptable excipients are available in the art, andinclude those listed in various pharmacopoeias. (See, e.g., USP, JP, EP,and BP, FDA web page (www.fda.gov), Inactive Ingredient Guide 1996, andHandbook of Pharmaceutical Additives, ed. Ash; Synapse InformationResources, Inc. 2002.)

Pharmaceutical dosage forms of a compound of the present invention maybe manufactured by any of the methods well-known in the art, such as,for example, by conventional mixing, sieving, dissolving, melting,granulating, dragee-making, tabletting, suspending, extruding,spray-drying, levigating, emulsifying, (nano/micro-) encapsulating,entrapping, or lyophilization processes. As noted above, thecompositions of the present invention can include one or morephysiologically acceptable inactive ingredients that facilitateprocessing of active molecules into preparations for pharmaceutical use.

Proper formulation is dependent upon the desired route ofadministration. For intravenous injection, for example, the compositionmay be formulated in aqueous solution, if necessary usingphysiologically compatible buffers, including, for example, phosphate,histidine, or citrate for adjustment of the formulation pH, and atonicity agent, such as, for example, sodium chloride or dextrose. Fortransmucosal or nasal administration, semisolid, liquid formulations, orpatches may be preferred, possibly containing penetration enhancers.Such penetrants are generally known in the art. For oral administration,the compounds can be formulated in liquid or solid dosage forms and asinstant or controlled/sustained release formulations. Suitable dosageforms for oral ingestion by a subject include tablets, pills, dragees,hard and soft shell capsules, liquids, gels, syrups, slurries,suspensions, and emulsions. The compounds may also be formulated inrectal compositions, such as suppositories or retention enemas, e.g.,containing conventional suppository bases such as cocoa butter or otherglycerides.

Solid oral dosage forms can be obtained using excipients, which mayinclude, fillers, disintegrants, binders (dry and wet), dissolutionretardants, lubricants, glidants, antiadherants, cationic exchangeresins, wetting agents, antioxidants, preservatives, coloring, andflavoring agents. These excipients can be of synthetic or naturalsource. Examples of such excipients include cellulose derivatives,citric acid, dicalcium phosphate, gelatine, magnesium carbonate,magnesium/sodium lauryl sulfate, mannitol, polyethylene glycol,polyvinyl pyrrolidone, silicates, silicium dioxide, sodium benzoate,sorbitol, starches, stearic acid or a salt thereof, sugars (i.e.dextrose, sucrose, lactose, etc.), talc, tragacanth mucilage, vegetableoils (hydrogenated), and waxes. Ethanol and water may serve asgranulation aides. In certain instances, coating of tablets with, forexample, a taste-masking film, a stomach acid resistant film, or arelease-retarding film is desirable. Natural and synthetic polymers, incombination with colorants, sugars, and organic solvents or water, areoften used to coat tablets, resulting in dragees. When a capsule ispreferred over a tablet, the drug powder, suspension, or solutionthereof can be delivered in a compatible hard or soft shell capsule.

In one embodiment, the compounds of the present invention can beadministered topically, such as through a skin patch, a semi-solid or aliquid formulation, for example a gel, a (micro-) emulsion, an ointment,a solution, a (nano/micro)-suspension, or a foam. The penetration of thedrug into the skin and underlying tissues can be regulated, for example,using penetration enhancers; the appropriate choice and combination oflipophilic, hydrophilic, and amphiphilic excipients, including water,organic solvents, waxes, oils, synthetic and natural polymers,surfactants, emulsifiers; by pH adjustment; and use of complexingagents. Other techniques, such as iontophoresis, may be used to regulateskin penetration of a compound of the invention. Transdermal or topicaladministration would be preferred, for example, in situations in whichlocal delivery with minimal systemic exposure is desired.

For administration by inhalation, or administration to the nose, thecompounds for use according to the present invention are convenientlydelivered in the form of a solution, suspension, emulsion, or semisolidaerosol from pressurized packs, or a nebuliser, usually with the use ofa propellant, e.g., halogenated carbons dervided from methan and ethan,carbon dioxide, or any other suitable gas. For topical aerosols,hydrocarbons like butane, isobutene, and pentane are useful. In the caseof a pressurized aerosol, the appropriate dosage unit may be determinedby providing a valve to deliver a metered amount. Capsules andcartridges of, for example, gelatin, for use in an inhaler orinsufflator, may be formulated. These typically contain a powder mix ofthe compound and a suitable powder base such as lactose or starch.

Compositions formulated for parenteral administration by injection areusually sterile and, can be presented in unit dosage forms, e.g., inampoules, syringes, injection pens, or in multi-dose containers, thelatter usually containing a preservative. The compositions may take suchforms as suspensions, solutions, or emulsions in oily or aqueousvehicles, and may contain formulatory agents, such as buffers, tonicityagents, viscosity enhancing agents, surfactants, suspending anddispersing agents, antioxidants, biocompatible polymers, chelatingagents, and preservatives. Depending on the injection site, the vehiclemay contain water, a synthetic or vegetable oil, and/or organicco-solvents. In certain instances, such as with a lyophilized product ora concentrate, the parenteral formulation would be reconstituted ordiluted prior to administration. Depot formulations, providingcontrolled or sustained release of a compound of the invention, mayinclude injectable suspensions of nano/micro particles or nano/micro ornon-micronized crystals. Polymers such as poly(lactic acid),poly(glycolic acid), or copolymers thereof, can serve ascontrolled/sustained release matrices, in addition to others well knownin the art. Other depot delivery systems may be presented in form ofimplants and pumps requiring incision.

Suitable carriers for intravenous injection for the molecules of theinvention are well-known in the art and include water-based solutionscontaining a base, such as, for example, sodium hydroxide, to form anionized compound, sucrose or sodium chloride as a tonicity agent, forexample, the buffer contains phosphate or histidine. Co-solvents, suchas, for example, polyethylene glycols, may be added. These water-basedsystems are effective at dissolving compounds of the invention andproduce low toxicity upon systemic administration. The proportions ofthe components of a solution system may be varied considerably, withoutdestroying solubility and toxicity characteristics. Furthermore, theidentity of the components may be varied. For example, low-toxicitysurfactants, such as polysorbates or poloxamers, may be used, as canpolyethylene glycol or other co-solvents, biocompatible polymers such aspolyvinyl pyrrolidone may be added, and other sugars and polyols maysubstitute for dextrose.

For composition useful for the present methods of treatment, atherapeutically effective dose can be estimated initially using avariety of techniques well-known in the art. Initial doses used inanimal studies may be based on effective concentrations established incell culture assays. Dosage ranges appropriate for human subjects can bedetermined, for example, using data obtained from animal studies andcell culture assays.

A therapeutically effective dose or amount of a compound, agent, or drugof the present invention refers to an amount or dose of the compound,agent, or drug that results in amelioration of symptoms or aprolongation of survival in a subject. Toxicity and therapeutic efficacyof such molecules can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., bydetermining the LD50 (the dose lethal to 50% of the population) and theED50 (the dose therapeutically effective in 50% of the population). Thedose ratio of toxic to therapeutic effects is the therapeutic index,which can be expressed as the ratio LD50/ED50. Agents that exhibit hightherapeutic indices are preferred.

The effective amount or therapeutically effective amount is the amountof the compound or pharmaceutical composition that will elicit thebiological or medical response of a tissue, system, animal, or humanthat is being sought by the researcher, veterinarian, medical doctor, orother clinician, e.g., reducing creatinine clearance, glomerularhyperfiltration and hyperperfusion, urine albumin excretion, ormicroalbuminuria, or treatment of early or late stage diabeticnephropathy, etc.

Dosages preferably fall within a range of circulating concentrationsthat includes the ED50 with little or no toxicity. Dosages may varywithin this range depending upon the dosage form employed and/or theroute of administration utilized. The exact formulation, route ofadministration, dosage, and dosage interval should be chosen accordingto methods known in the art, in view of the specifics of a subject'scondition.

Dosage amount and interval may be adjusted individually to provideplasma levels of the active moiety that are sufficient to achieve thedesired effects, e.g., regulation of glucose metabolism, decrease inblood glucose levels, etc., i.e., minimal effective concentration (MEC).The MEC will vary for each compound but can be estimated from, forexample, in vitro data and animal experiments. Dosages necessary toachieve the MEC will depend on individual characteristics and route ofadministration. In cases of local administration or selective uptake,the effective local concentration of the drug may not be related toplasma concentration.

The amount of agent or composition administered may be dependent on avariety of factors, including the sex, age, and weight of the subjectbeing treated, the severity of the affliction, the manner ofadministration, and the judgment of the prescribing physician.

The present compositions may, if desired, be presented in a pack ordispenser device containing one or more unit dosage forms containing theactive ingredient. Such a pack or device may, for example, comprisemetal or plastic foil, such as a blister pack, or glass and rubberstoppers such as in vials. The pack or dispenser device may beaccompanied by instructions for administration. Compositions comprisinga compound of the invention formulated in a compatible pharmaceuticalcarrier may also be prepared, placed in an appropriate container, andlabeled for treatment of an indicated condition.

These and other embodiments of the present invention will readily occurto those of ordinary skill in the art in view of the disclosure herein.

EXAMPLES

The invention will be further understood by reference to the followingexamples, which are intended to be purely exemplary of the invention.These examples are provided solely to illustrate the claimed invention.The present invention is not limited in scope by the exemplifiedembodiments, which are intended as illustrations of single aspects ofthe invention only. Any methods which are functionally equivalent arewithin the scope of the invention. Various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

Example 1 Treatment of Early Stage Features of Diabetic Nephropathy

The methods of the invention were used to demonstrate broad-spectrumefficacy in an animal model for certain aspects of early stage diabeticnephropathy as follows. Eight-week-old mice having a loss-of-functionmutation in the leptin receptor (Ob-R; encoded by the db gene) wereobtained from Harlan, Indianapolis Ind. These db/db mice serve as ananimal model of obese type 2 diabetes, and, in particular, a model ofobese type 2 diabetic nephropathy characterized by early aspects ofdiabetic nephropathy, including, for example, kidney hyperfiltration andproteinuria with minimal development of interstitial fibrosis. This isan animal model of early stage diabetic nephropathy rather than latestage diabetic nephropathy, as evidenced by the minimal development ofinterstitial fibrosis. Homozygous db/db (diabetic) are hyperglycemic at8 weeks of age. Homozygous db/db (diabetic) and heterozygousdb/+(non-diabetic) animals were treated (intraperitoneal injection) witheither anti-CTGF monoclonal antibody (αCTGF) (prepared as described inInternational Publication No. WO 2004/108764 or by the cell lineidentified by ATCC Accession No. PTA-6006, deposited 20 May 2004) orcontrol human IgG (cIgG). In all animals, an initial injection of 300 μgof antibody was followed by 100 μg doses administered 3 times per weekfor 60 days. Blood samples were collected and body weights were measuredat the beginning of and periodically throughout the treatment period.Food consumption was also recorded.

Table 1 below shows the mean body weight (BW), blood glucose level (BG),and food consumption (FC) at day 0 and day 60 in cIgG-treated db/+mice,αCTGF-treated dbl+mice, cIgG-treated diabetic db/db mice, andaCTGF-treated db/db mice. All data are expressed as Mean±SEM. The numberof mice per group (n) ranged from 9 to 15. Non-diabetic (db/+) animalsthat presented with polycystic kidneys were excluded from the analysis.As shown in Table 1, a clear distinction existed between the diabetic(db/db) animals and the non-diabetic (dbl+) animals with respect to bodyweight, blood glucose levels, and food consumption. Treatment witheither anti-CTGF antibody or cIgG did not significantly affect overallweight gain, blood glucose levels, or food consumption in eitherdiabetic (db/db) or non-diabetic (db/+) animals. TABLE 1 Day 0 Day 60Group, Treatment BW BG FC BW BG FC (no. of animals) (g) (mM) (g/24 h)(g) (mM) (g/24 h) db/+, cIgG 20.6 ± 0.3  6.3 ± 0.2 4.8 ± 0.1 22.4 ± 0.4 6.0 ± 0.2 5.0 ± 0.1 (n = 11) db/+, αCTGF 20.2 ± 0.4  6.1 ± 0.2 4.7 ± 0.221.6 ± 0.3  6.2 ± 0.1 5.1 ± 0.2 (n = 9) db/db, cIgG 39.4 ± 0.6* 16.7 ±1.0* 10.0 ± 0.2* 47.1 ± 1.0* 22.1 ± 0.8* 10.8 ± 0.2* (n = 15) db/db,αCTGF 38.0 ± 1.0* 15.8 ± 0.7* 10.2 ± 0.1* 47.7 ± 1.2* 21.7 ± 0.8* 10.5 ±0.1* (n = 14)Data are expressed as mean ± SEM.*P < 0.01 vs. db/+mice.Inhibition of Progression of Early Stage Features of DiabeticNephropathy

Following the anti-CTGF antibody treatment period described above,various measurements of kidney function and nephropathy were obtained,including kidney weight, creatinine clearance, urinary albuminexcretion, and urine volume. Table 2 below shows the mean kidney weight(KW), creatinine clearance (CrCl), and 24-hour urinary albumin excretion(UAE) at day 60 in cIgG-treated dbl+mice, αCTGF-treated db/+mice,cIgG-treated db/db mice, and αCTGF-treated db/db mice. All data areexpressed as Mean±SEM. The number of mice per group (n) ranged from 9 to15. As stated above, non-diabetic (db/+) animals that presented withpolycystic kidneys were excluded from the analysis. TABLE 2 Group,Treatment KW CrCl UAE (no. of animals) (mg) (ml/h) (μg/24 h) db/+, cIgG133.8 ± 5.1 2.17 ± 0.29 0.30 ± 0.02 (n = 11) db/+, αCTGF 141.0 ± 4.32.37 ± 0.19 0.23 ± 0.04 (n = 9) db/db, cIgG 207.8 ± 3.9** 5.39 ± 0.36**2.52 ± 0.20** (n = 15) db/db, αCTGF 177.4 ± 4.5* 2.76 ± 0.31^(Δ) 0.98 ±0.09^(□) (n = 14)Data are expressed as mean ± SEM.**P < 0.01 vs. db/+ mice.*P < 0.01 vs. db/+ mice and P < 0.05 cIgG-treated db/db mice.^(Δ)P < 0.01 vs. cIgG-treated db/db mice.^(□)P < 0.01 vs. db/+ mice and cIgG-treated db/db mice.

As shown in Table 2, db/db mice exhibited hyperfunctioning kidneys asindicated by renal enlargement (i.e., increased kidney weight) (FIG. 1),increased creatinine clearance (FIG. 2), and increased urinary albuminexcretion (FIG. 3, *P<0.01 vs. anti-CTGF-treated db/+). Diabetic animalstreated with anti-CTGF antibody showed reduced kidney weight gaincompared to diabetic animals treated with cIgG.

Creatinine clearance in cIgG-treated db/db animals was approximatelytwice the value observed in db/+animals, indicating impaired renalfunction, hypertrophy, and hyperfiltration in diabetic animals. Theurinary albumin excretion was also increased in diabetic db/db animalscompared to the urinary albumin excretion observed in non-diabeticdbl+animals. The db/db animals treated with anti-CTGF antibody hadcreatinine clearance and urinary albumin excretion levels markedly lowerthan those observed in the cIgG-treated db/db animals. Specifically, theanti-CTGF treated diabetic mice had creatinine clearance levels 82%below that seen in the cIgG-treated diabetic mice. The anti-CTGF treateddiabetic mice had urinary albumin excretion levels 69% below that seenin the cIgG-treated diabetic mice. These results provide evidence of adramatic improvement in kidney function in the anti-CTGF antibodytreated mice. Treatment of non-diabetic animals using methods of theinvention showed no adverse effects on kidney weight or function. Thesedata showed that administration of anti-CTGF antibody to diabeticanimals resulted in reduced kidney weight gain, creatinine clearance,and urinary albumin excretion.

Further, diabetic (db/db) mice showed increased urine volume compared tonon-diabetic-(db/+) mice. Administration of anti-CTGF antibody asdescribed above reduced urine volume in diabetic (db/db) mice. (See FIG.5, *P<0.01 vs. anti-CTGF-treated db/+) This data indicated thatadministration of anti-CTGF antibody to diabetic animals reduced urinaryvolume. These results also indicated that inhibition of CTGF provides amethod for reducing increased urinary volume associated with diabeticnephropathy, and therefore provides a method for improving kidneyfunction.

Analysis of changes in glomerular volume (e.g., reduction in glomerularvolume expansion) and basement membrane thickening further demonstratedthe efficacy of inhibition of CTGF in treating and preventing thedevelopment and progression of diabetic nephropathy. As shown in FIG. 6(*not different from anti-CTGF-treated db/+), treatment of diabetic(db/db) animals with anti-CTGF antibody reduced basement membranethickening.

Taken together, these data showed that treatment of diabetic (db/db)animals with anti-CTGF antibody reduced kidney hypertrophy (as evidencedby lower kidney weight in anti-CTGF treated diabetic animals) andrestored kidney function (as evidenced by a reduced increase increatinine clearance and urinary excretion rate in anti-CTGF treateddiabetic animals). These results also indicated that inhibition of CTGFprovided a method for reducing glomerular permeability andhyperfiltration, as well as reducing mesangial expansion and basementmembrane thickening. Therefore, inhibition of CTGF provides atherapeutic approach for treating early stage features of diabeticnephropathy.

Example 2 CTGF Implicated in Early Stage Features of ProgressiveVitreoretinal Disorders

The association between CTGF and ocular disease, including retinaldisorders, has been previously established. (See, e.g., InternationalPublication No. WO 03/049773.) Here, the relationship between ocularconcentrations of CTGF and VEGF and the degree of neovascularization andfibrosis were examined to determine the correlation, if any, betweenCTGF and VEGF expression in vitreous. A correlation would be suggestiveof CTGF involvement in both early stage and late stage aspects of oculardisorders. Undiluted vitreous samples (0.5 to 1 ml) were obtained at thestart of a pars plana vitrectomy in patients with proliferativevitreoretinopathy (PVR), proliferative diabetic retinopathy (PDR),macular pucker, or macular hole. Samples of vitreous fluids werecollected in sterile tubes, immediately frozen in dry ice, and stored at−80° C. until assayed for CTGF and VEGF.

Neovascularization associated with all retinal disorders was graded asfollows: grade 0, no neovascularization; grade 1, quiescentneovascularization, with only non-perfused, gliotic vessels present; andgrade 2, active neovascularization, with perfused preretinalcapillaries. (See Aiello et al. (1994) N Engl J. Med 331:1480-1487.)

CTGF and VEGF levels in vitreous samples were measured by ELISA.Briefly, vitreous samples were centrifuged at 14,000 rpm for 15 minutesat 4° C. and the supernatant collected. CTGF levels were measured bysandwich ELISA using two monoclonal antibodies to human CTGF, each ofwhich specifically recognizes a distinct region of the N-terminalportion of CTGF as follows. Microtiter plates were coated overnight at4° C. with capture anti-CTGF monoclonal antibody (10 μg/ml) in coatingbuffer (50 mM sodium borate, pH 9.6). The plates were blocked with 100μl 1% BSA in phosphate buffered saline for 2 hours at room temperatureand then washed with wash buffer (phosphate buffered saline containing0.05% Tween 20). Vitreous samples were diluted 5 times in assay buffer(50 mM TRIS, pH 7.7, 0.1% BSA, 4 mM MgCl₂, 400 mM ZnCl₂, 0.05% NaN₃, 50mg/L sodium heparin, and 0.1% Triton X-100). To each well was added 50μl of diluted vitreous sample together with 50 mM of biotinylatedmonoclonal anti-human CTGF detection antibody (diluted in assay buffer).The plates were incubated for 2 hours at 37° C., washed with washbuffer, and incubated with 100 μl/well streptavidin-conjugated alkalinephosphatase (1 μg/ml diluted in assay buffer) (Jackson ImmunoresearchLaboratories) for 1 hour at room temperature. Following this incubation,the plates were washed with wash buffer and 100 μl of substrate solution(1 mg/ml, p-nitrophenyl phosphate, Sigma Chemical Co.) in diethanolaminebuffer (1 M diethanolamine, 0.5 mM MgCl₂, 0.02% NaN₃, pH 9.8) was addedto each well. Absorbance was read at 405 nm on a Bio-Rad microplatereader. Purified recombinant human CTGF was used as a standard. Vitreouslevels of VEGF-165 were determined by a commercially available sandwichELISA according to the manufacturer's instructions (R&D Systems).

A significant positive correlation between CTGF levels and the degree ofneovascularization was observed (p=0.0153). As shown in Table 3 below,patients with the highest degree (grade 2) of neovascularization hadsignificantly higher CTGF levels than patients with a lower degree(grade 0 or grade 1) of neovascularization. FIG. 4 shows intravitrealCTGF and VEGF levels were positively correlated (r=0.544, p=0.001).Significantly, these results demonstrate for the first time a directcorrelation between CTGF and VEGF levels measured from the same sample,at the same stage of disease. TABLE 3 Neovascularization Grade CTGFLevels 95% CI Grade 0  8.7 ng/ml  6.9-11.0 Grade 1 14.0 ng/ml 11.2-17.5Grade 2 20.8 ng.ml 16.2-26.9

Results from the experiments described above showed that CTGF is presentin human vitreous and its concentration significantly and stronglycorrelated with the presence and degree of neovascularization and thatvitreous levels of CTGF strongly correlated with vitreous levels ofVEGF. While it has been established that CTGF is associated with thedevelopment and progression of ocular fibrosis and other late-stageaspects of retinal disease, the present results taken together with theresults shown in Example 1 and Example 3 herein, implicate CTGF as wellas VEGF as a critical factor in early stage development of progressivedisease, including diabetic nephropathy and various vitreoretinaldisorders. Therefore, the present invention provides methods fortreating both early (e.g., neovascularization) and late (e.g., fibrosis)stages of retinopathies, such as, PVR, PDR, etc.

Example 3 Treatment of Late Stage Features of Diabetic Nephropathy

The effect of anti-CTGF therapy was examined in an animal model of latestage diabetic nephropathy. As has been previously described using thisanimal model, rats with diabetes mellitus exhibited high susceptibilityto unilateral renal ischemia reperfusion, resulting in rapidlyprogressive nephropathy and end-stage renal failure, associated withdevelopment of fibrosis, atrophy of the kidney, and severely compromisedglomerular filtration rate. (See, e.g., Melin et al. (1997) Kidney Int52:985-991.) In this animal model of diabetes mellitus, ischemiaseverely impaired kidney function in diabetic rats. In this animalmodel, the renal effects on kidney function and pathology ofhyperglycemia and ischemia are similar to those observed in human latestage diabetic nephropathy and end-stage renal disease (ESRD).

Diabetes mellitus was induced in male Sprague Dawley rats by a singlei.v. dose of streptozotocin (STZ) (50 mg/kg). Unilateral renal ischemiareperfusion (1R) was achieved in one kidney by clamping the left renalartery for 30 minutes, thereby preventing blood flow to the left kidney.Treatment with anti-CTGF monoclonal antibody (i.p. 5 mg/kg) wasinitiated 1 day before renal ischemia reperfusion (i.e., 2 weeks afterthe development of diabetes) and continued 3 times per week for 10weeks. Control animals not receiving anti-CTGF antibody wereadministered PBS (i.p. 5 ml/kg). Blood samples were obtained from thetail vein. Blood clinical chemistry, performed by Quality Clinical Labs,Inc. (Mountain View, Calif.), was analyzed at weeks 0, 4, 8, and 10.Total 24-hour urinary protein was determined at weeks 5 and 9.Individual rats were placed in metabolic cages and 24-hour urinespecimens were collected. Urine volume was measured and urine proteinwas analyzed using a BCA Protein Assay Kit (Pierce Chemical Co.).

Glomerular filtration rate (GFR) is the most widely measurement ofkidney function. Inulin clearance is a measurement of glomerularfiltration rate. In these experiments, glomerular filtration rate (e.g.,kidney function) was determined for individual kidneys by measurement ofurine volume and inulin clearance. Urine was collected via a cannulatedureter and blood was collected from the femoral artery. Urine volume wasestimated gravimetrically. Inulin concentration was determined using theAnthrone method. Inulin clearance, indicative of GFR, was determinedusing the formula: (U_(cone)×U_(vol))/S_(cone). At the end of theexperiment, kidneys were removed for biochemical and histopathologicalevaluation.

Data are presented as mean+/−SEM. Data were compared within theexperimental groups at each time point using one-way analysis ofvariance (ANOVA) and Student-Newman-Keuls method (SIGMASTAT). When onlytwo groups were compared, a t-Test was used (Two-Sample Assuming EqualVariances analysis tool, Microsoft Excel). A value of P<0.05 wasconsidered significant.

Animals administered a single dose of STZ became diabetic, as indicatedby elevated blood glucose levels. Blood glucose levels increased fromless than 200 mg/dL in control (non-STZ-treated) animals to levelsgreater than 600 mg/dL in STZ-treated animals, indicating that theseanimals were diabetic. Renal IR of non-diabetic (i.e., non-STZ-treated)animals did not increase blood glucose levels above that of controlanimals (data not shown). Blood glucose levels remained elevated inSTZ-treated animals throughout the 10 weeks following unilateral renalIR. (Data not shown.).

Late Stage Proteinuria

Microalbuminuria characteristic of early stage diabetic nephropathyprogresses to macroalbuminuria and late stage proteinuria. In animalswith diabetes mellitus, significant increases in 24-hour total urinaryprotein (i.e., late stage proteinuria) were observed, indicatingincreased glomerular hyperfiltration and development of renal failure.As shown in FIG. 7, total urine protein in non-diabetic animals(sham+PBS; IR+PBS) was approximately 100 mg/24-hours. (In FIG. 7,*higher than non-diabetic (p<0.001), # lower than DM+IR+PBS (p<0.05) atcorresponding weeks.) Diabetic animals with renal IR, however, had totalurine protein levels exceeding 350 mg/24-hours. Administration ofanti-CTGF antibody to diabetic animals with renal IR resulted in asignificant reduction in 24-hour total urine protein at weeks 5 and 9,to approximately 225 mg/24-hours and 250 mg/24-hours, respectively,compared to non-treated diabetic animals. (See FIG. 7.) This data showedthat administration of an antibody to CTGF reduced proteinuria indiabetic animals. These results indicated that inhibition of CTGFprovides a therapeutic means for decreasing kidney hyperfiltration.These results demonstrate for the first time that anti-CTGF therapy isuseful for preventing the development and progression of late stageproteinuria.

Blood Urea-Nitrogen (BUN)

Increased BUN levels are indicative of impaired kidney functionassociated with late stage diabetic nephropathy. Significant increasesin BUN levels were observed in these diabetic animals. BUN levels incontrol non-diabetic animals (sham+PBS; IR+PBS) were below 20 mg/dL at0, 4, and 10 weeks of the study. In diabetic animals with renal IR, BUNlevels increased from approximately 22 mg/dL at week zero, to greaterthan 40 mg/dL at 4 weeks. (See FIG. 8, *higher than sham+PBS and IR+PBS(p<0.01), # lower than DM+IR+PBS (p<0.01) at 4 weeks.) Administration ofanti-CTGF monoclonal antibody to diabetic animals with renal IR resultedin a reduction of BUN levels at 4 weeks (to approximately 30 mg/dL) andat 10 weeks (to approximately 35 mg/dL), compared to that observed indiabetic animals without anti-CTGF antibody administration. (See FIG.8.) These results showed for the first time that anti-CTGF therapy isuseful for reducing BUN levels in diabetics, indicating that thatinhibition of CTGF provides a therapeutic approach for improving kidneyfunction.

Glomerular Filtration Rate

Glomerular filtration rate was determined for individual kidneys foreach of the various experimental conditions described above. In controlanimals (i.e., non-diabetic, non-IR), GFR was greater than 0.3mL/min/kidney/100 g. Non-diabetic animals with renal IR had a GFR ofapproximately 0.28 mL/min/kidney/100 g. Diabetic animals without renalIR had a GFR of approximately 0.17 mL/min/kidney/100 g. (Data notshown.)

Glomerular filtration rate was drastically reduced in the ischemickidney of animals with diabetes mellitus at 10 weeks, to approximately0.01 mL/min/kidney/100 g. (See FIG. 9.) Administration of anti-CTGFantibody significantly improved glomerular filtration rate in individualkidneys in diabetic animals affected by renal IR, to a level greaterthan 0.035 mL/min/kidney/100 g. This data showed that administration ofan antibody to CTGF increased glomerular filtration rate in diabeticanimals in late-stage renal disease. These results demonstrated for thefirst time that anti-CTGF therapy is effective at improving glomerularfiltration rate in late stage diabetic nephropathy, and therefore,provides a therapeutic approach for improving kidney function inlate-stage renal disease.

Various modifications of the invention, in addition to those shown anddescribed herein, will become apparent to those skilled in the art fromthe foregoing description. Such modifications are intended to fallwithin the scope of the appended claims.

All references cited herein are hereby incorporated by reference hereinin their entirety.

1. A method for reducing creatinine clearance in a subject having or atrisk for having diabetes or early stage diabetic nephropathy, the methodcomprising administering to the subject a therapeutically effectiveamount of an agent that inhibits CTGF.
 2. A method for reducingglomerular hyperfiltration in a subject having or at risk for havingdiabetes or early stage diabetic nephropathy, the method comprisingadministering to the subject a therapeutically effective amount of anagent that inhibits CTGF.
 3. A method for reducing glomerularhyperperfusion in a subject having or at risk for having diabetes orearly stage diabetic nephropathy, the method comprising administering tothe subject a therapeutically effective amount of an agent that inhibitsCTGF.
 4. A method for reducing urinary albumin excretion in a subjecthaving or at risk for having diabetes or diabetic nephropathy, themethod comprising administering to the subject a therapeuticallyeffective amount of an agent that inhibits CTGF.
 5. A method forreducing or preventing kidney weight gain in a subject having or at riskfor having diabetes or diabetic nephropathy, the method comprisingadministering to the subject a therapeutically effective amount of anagent that inhibits CTGF.
 6. A method for normalizing glomerularfiltration rate in a subject having or at risk for having diabetes ordiabetic nephropathy, the method comprising administering to the subjecta therapeutically effective amount of an agent that inhibits CTGF.
 7. Amethod for reducing glomerular hypertrophy in a subject having or atrisk for having diabetes or diabetic nephropathy, the method comprisingadministering to the subject a therapeutically effective amount of anagent that inhibits CTGF.
 8. A method for reducing proteinuria in asubject having or at risk for having diabetes or diabetic nephropathy,the method comprising administering to the subject a therapeuticallyeffective amount of an agent that inhibits CTGF.
 9. A method forreducing albuminuria in a subject having or at risk for having diabetesor diabetic nephropathy, the method comprising administering to thesubject a therapeutically effective amount of an agent that inhibitsCTGF.
 10. A method for reducing microalbuminuria in a subject having orat risk for having diabetes or diabetic nephropathy, the methodcomprising administering to the subject a therapeutically effectiveamount of an agent that inhibits CTGF.
 11. A method for reducingmacroalbuminuria in a subject having or at risk for having diabetes ordiabetic nephropathy, the method comprising administering to the subjecta therapeutically effective amount of an agent that inhibits CTGF.
 12. Amethod for reducing BUN levels in a subject having or at risk for havingdiabetes or diabetic nephropathy, the method comprising administering tothe subject a therapeutically effective amount of an agent that inhibitsCTGF.
 13. A method for reducing inulin clearance in a subject having orat risk for having diabetes or diabetic nephropathy, the methodcomprising administering to the subject a therapeutically effectiveamount of an agent that inhibits CTGF.
 14. A method for preventing,reducing the risk of, or delaying the onset of diabetic complications ina subject at risk for developing such complications, the methodcomprising administering to the subject a therapeutically effectiveamount of an agent that inhibits CTGF.
 15. A method for treatingincipient diabetic nephropathy in a subject having or at risk for havingincipient diabetic nephropathy, the method comprising administering tothe subject a therapeutically effective amount of an agent that inhibitsCTGF.
 16. A method for treating incipient diabetic nephropathy in asubject having or at risk for having early stage diabetic nephropathy,the method comprising administering to the subject a therapeuticallyeffective amount of an agent that inhibits CTGF.
 17. A method fortreating overt diabetic nephropathy in a subject having or at risk forhaving overt diabetic nephropathy, the method comprising administeringto the subject a therapeutically effective amount of an agent thatinhibits CTGF.
 18. A method for treating diabetic nephropathy in asubject having or at risk for having diabetic nephropathy, the methodcomprising administering to the subject a therapeutically effectiveamount of an agent that inhibits CTGF in combination with an inhibitingamount of an angiotensin converting enzyme inhibitor.
 19. A method fortreating diabetic nephropathy in a subject having or at risk for havingdiabetic nephropathy, the method comprising administering to the subjecta therapeutically effective amount of an agent that inhibits CTGF incombination with an inhibiting amount of an angiotensin receptorblocker.
 20. A method for treating or preventing early stage aspects ofa progressive disease in a subject having or at risk for having such adisease, the method comprising administering to the subject atherapeutically effective amount of an agent that inhibits CTGF.
 21. Amethod of treating progressive renal failure in a subject, the methodcomprising administering to the subject a therapeutically effectiveamount of an agent that inhibits CTGF.
 22. A method for improving kidneyfunction in a subject having or at risk for having impaired kidneyfunction, the method comprising administering to the subject atherapeutically effective amount of a an agent that inhibits CTGF.